Emerging knowledge of regulatory roles of <Emphasis Type="SmallCaps">d-</Emphasis>amino acids in bacteria

The d-enantiomers of amino acids have been thought to have relatively minor functions in biological processes. While l-amino acids clearly predominate in nature, d-amino acids are sometimes found in proteins that are not synthesized by ribosomes, and d-Ala and d-Glu are routinely found in the peptidoglycan cell wall of bacteria. Here, we review recent findings showing that d-amino acids have previously unappreciated regulatory roles in the bacterial kingdom. Many diverse bacterial phyla synthesize and release d-amino acids, including d-Met and d-Leu, which were not previously known to be made. These noncanonical d-amino acids regulate cell wall remodeling in stationary phase and cause biofilm dispersal in aging bacterial communities. Elucidating the mechanisms by which d-amino acids govern cell wall remodeling and biofilm disassembly will undoubtedly reveal new paradigms for understanding how extracytoplasmic processes are regulated as well as lead to development of novel therapeutics.     

Free amino acid composition of the intestinal contents,intestinal cells and hemolymph ofPhilosamia cynthia larvae

Summary Free amino acid composition of the intestinal contents, intestinal cells and hemolymph has been determined in larvae of the mothPhilosamia cynthia. From the hemolymph/lumen concentration ratio, an active transport could be inferred for neutral and basic amino acids. The values of cell/lumen and hemolymph/cell ratios suggested that the active step in the transport mechanism could be localized at the luminal pole of the enterocyte for neutral amino acids (except aromatic amino acids) and at the basolateral pole of the enterocyte for basic amino acids (except arginine).This work was supported by grants from Italian Consiglio Nazionale delle Ricerche and from Ministero della Pubblica Istruzione, Rome. The authors are indebted to Prof. V. Capraro for helpful discussion.     

Control of cell growth: Rag GTPases in activation of TORC1

The target of rapamycin (TOR) is a central regulator controlling cell growth. TOR is highly conserved from yeast to mammals, and is deregulated in human cancers and diabetes. TOR complex 1 (TORC1) integrates signals from growth factors, cellular energy status, stress, and amino acids to control cell growth, mitochondrial metabolism, and lipid biosynthesis. The mechanisms of growth factors and cellular energy status in regulating TORC1 have been well established, whereas the mechanism by which amino acid induces TORC1 remains largely unknown. Recent studies revealed that Rag GTPases play a central role in the regulation of TORC1 activation in response to amino acids. In this review, we will discuss the recent progress in our understanding of Rag GTPase-regulated TORC1 activation in response to amino acids. Particular focus will be given to the function of Rag GTPases in TORC1 activation and how Rag GTPases are regulated by amino acids.     

Synthesis of three NH2-terminally extended arginine-vasopressins with prolonged biological activities

The synthesis of three novel AVP-analogues, extended by 1-3 amino acids at their NH2-2-termini in accordance with the sequence of the bovine arginine-vasopressin neurophysin II precursor, is reported. The compounds were assayed for their antidiuretic and vasopressor activities with particular attention to the duration of the effects. All compounds showed high potency, based on the intensity, and prolonged effects in both test systems compared with AVP.     

Inhibition of arginase in sheep brain homogenates by some L-amino acids

Summary The effect of 16 L-amino acids on the activity levels of arginase in sheep brain homogenates was studied. The amino acids leucine, valine, lysine, and ornithine inhibited arginase activity significantly. The other amino acids tested did not show a significant influence on arginase activity. The inhibition was related to the carbon chain length of the amino acids.     

Covalent immobilization as a stimulus of cell wall composition changes

Covalent immobilization of yeast cells by an activated diamine spacer is accompanied by increased levels of cell wall proteins, lipids, amino sugars, amino acids and acid phosphatase leakage, and by altered composition of mannoproteins. The observed changes in cell wall composition are attributed to the effect of cell-solid surface contact.     

Fungitoxicity ofm-fluorophenylalanine-containing peptides towardsPythium ultimum

Summary The tripeptide L-m-fluorophenylalanyl-L-alanyl-L-alanine was much more fungitoxic towardsPythium ultimum than the dipeptide L-m-fluorophenylalanyl-L-alanine orm-fluorophenylalanine. The fungitoxicity of the tripeptide was reduced by L-alanyl peptides and phenylalanine, but not by other amino acids. In contrast, the fungitoxicity ofm-fluorophenylalanine was unaffected by peptides, and was antagonized by several amino acids. These results suggest the effective delivery ofm-fluorophenylalanine into the cell by a tripeptide carrier.     

Tetrapyrrole biosynthesis from 4,5-dioxovaleric acid in rhodopseudomonas spheroides.

Prophyrin biosynthesis from 4,5-dioxovaleric acid was studied in cell suspensions of R. spheroides. The experiments show that 4,5-dioxovaleric acid is a far precursor of porphyrins through delta amino laevulinic acid formation in a transmination reaction involving also l-alanine. It differs radically from the classical delta aminolaevulinic acid synthesis using glycine and succinyl CoA as substrates.     

Synthesis, structure and function of poly-α-amino acids – the simplest of protein models

During the 1950s, linear and multichain poly-α-amino acids were synthesized by polymerization of the corresponding N-carboxyamino acid anhydrides in solution in the presence of suitable catalysts. The resulting homo- and heteropolymers have since been widely employed as simple protein models. Under appropriate conditions, poly-α-amino acids, in the solid state and in solution, were found to acquire conformations of an α-helix and of β-parallel and antiparallel pleated sheets, or to exist as random coils. Their use in experimental and theoretical investigations of helix-coil transitions helped to shed new light on the mechanisms involved in protein denaturation. Conformational fluctuations of peptides in solution were analysed theoretically and studied experimentally by nonradiative energy-transfer techniques. Poly-α-amino acids played an important role in the deciphering of the genetic code. In addition, analysis of the antigenicity of poly-α-amino acids led to the elucidation of the factors determining the antigenicity of proteins and peptides. The synthetic procedures developed made possible the preparation of immobilized enzymes which were shown to be of considerable use as heterogeneous biocatalysts in the chemical and pharmaceutical industry. Interest in the biological and physicochemical characteristics of poly-α-amino acids was recently renewed because of the reported novel findings that some copolymers of amino acids are effective as drugs in multiple sclerosis, and that glutamine repeats and reiteration of other amino acids occur in inherited neurodegenerative diseases.     

Ageing of the erythrocyte. XVI. Free amino acid content

Summary The concentrations of some free amino acids, especially glycine, histidine, isoleucine, methionine, phenylalanine, serine and tyrosine decrease considerably during the intravascular ageing of bovine erythrocytes. They seem to provide as sensitive an indication of red cell age as creatine does.Stimulating discussions with Prof. W. Leyko and the experimental help of Mrs A. Rogala, are greatly acknowledged.     

Synphilin-1 inhibits alpha-synuclein degradation by the proteasome

Intracellular deposits of aggregated alpha-synuclein are a hallmark of Parkinson’s disease. Protein–protein interactions are critical in the regulation of cell proteostasis. Synphilin-1 interacts both in vitro and in vivo with alpha-synuclein promoting its aggregation. We report here that synphilin-1 specifically inhibits the degradation of alpha-synuclein wild-type and its missense mutants by the 20S proteasome due at least in part by the interaction of the ankyrin and coiled-coil domains of synphilin-1 (amino acids 331–555) with the N-terminal region (amino acids 1–60) of alpha-synuclein. Co-expression of synphilin-1 and alpha-synuclein wild-type in HeLa and N2A cells produces a specific increase in the half-life of alpha-synuclein, as degradation of unstable fluorescent reporters is not affected. Synphilin-1 inhibition can be relieved by co-expression of Siah-1 that targets synphilin-1 to degradation. Synphilin-1 inhibition of the proteasomal pathway of degradation of alpha-synuclein may help to understand the pathophysiological changes occurring in PD and other synucleinopathies.     

Uptake of L-glutamate and L-aspartate in neurones and glial cells of cultured human and rat spinal cord.

Autoradiographic investigations on the uptake of L-glutamate and L-aspartate have shown that the amino acids were taken up by neurones as well as by glial cells of cultured human and rat spinal cord. The activity of glutamate and aspartate varied considerably between individual neurones, whereas glial cells showed a more even distribution of the labelled amino acids. Our results suggest that both neurones and glial cells are involved in the uptake of amino acid transmitters.     

Fungitoxicity of m-fluorophenylalanine-containing peptides towards Pythium ultimum

The tripeptide L-m-fluorophenylalanyl-L-alanyl-L-alanine was much more fungitoxic towards Pythium ultimum than the dipeptide L-m-fluorophenylalanyl-L-alanine or m-fluorophenylalanine. The fungitoxicity of the tripeptide was reduced by L-alanyl peptides and phenylalanine, but not by other amino acids. In contrast, the fungitoxicity of m-fluorophenylalanine was unaffected by peptides, and was antagonized by several amino acids. These results suggest the effective delivery of m-fluorophenylalanine into the cell by a tripeptide carrier.     

Changes in the free amino acids of the haemolymph of diapause and non-diapause pupae of the cotton bollworm,Heliothis armigera Hbn. (Lepidoptera: Noctuidae)

Summary The concentration of free amino acids in the haemolymph of non-diapausing and diapausing pupae ofHeliothis armigera was investigated. 20 amino acids were detected in the haemolymph of the studied stages. Asparagine, glutamine, cystine, ornithine, histidine and valine were the predominant free amino acids at all stages. The diapause resulted in increased levels of most of the amino acids.     

Conformational constraints on side chains in protein residues increase their information content

Like all other complex biological systems, proteins exhibit properties not seen in free amino acids (i.e., emergent properties). The present investigation arose from the deduction that proteins should offer a good model to approach the reverse phenomenon, namely top-down constraints experienced by protein residues compared to free amino acids. The crystalline structure of profilin Ib, a contractile protein of Acanthamoeba castellanii, was chosen as the object of study and submitted to 2-ns molecular dynamics simulation. The results revealed strong conformational constraints on the side chain of residues compared to the respective free amino acids. A Shannon entropy (SE) analysis of the conformational behavior of the side chains showed in most cases a strong decrease in the SE of the 1 and 2 dihedral angles compared to free amino acids. This is equivalent to stating that conformational constraints on the side chain of residues increase their information content and hence recognition specificity compared to free amino acids. In other words, the vastly increased information content of a protein relative to its free monomers is embedded not only in the tertiary structure of the backbone, but also in the conformational behavior of the side chains. The postulated implication is that both backbone and side chains, by virtue of being conformationally constrained, contribute to the recognition specificity of the protein toward other macromolecules and ligands.Received 13 July 2003; received after revision 18 August 2003; accepted 4 September 2003     

Reevaluation of hydropathy profiles of voltage-gated ionic channels

A reevaluation of the secondary structure of Na, Ca and K channel proteins led to the following results. Only three segments (S1, S5 and S6) of each repeat are sufficiently hydrophobic to be predicted as transmembrane helices, if a window of 19 amino acids is used. Some of the S2 and S3 segments show higher hydrophobic values when calculated with the window of 9 amino acids and can be predicted as short helices. S4 segments are strongly hydrophilic and cannot be predicted as transmembrane helices. Some of the S2, S3 and S4 segments have an amphipathic character; however, these helices do not span a membrane. A model is proposed where 12 hydrophobic transmembrane helices surround 12 shorter helices, forming a hydrophilic pore. In addition, a unique pattern for S4 segments of voltage-gated channel proteins is defined.     

Selective antagonism by Mg2+ of amino acid-induced depolarization of spinal neurones.

In the isolated frog or rat spinal cord, low concentrations of Mg2+ (0.5-1.00 mM) markedly depress, in a substantially Ca2+-independent manner, ventral root depolarizations produced by dorsal root stimulation and by certain amino acids (e.g. N-methyl-D-aspartate and L-homocysteate) but do not depress depolarizations produced by other excitatory amino acids (e.g. kainate and quisqualate). L-Aspartate-induced depolarizations are more sensitive to Mg2+ then are L-glutamate-induced depolarizations.     

Effect of creatine and other phosphorylable amino compounds on the ileal absorption of Ca-45 in the rat]

Some carbohydrates which increase calcium absorption were phosphate acceptors. When administrated to adult Rat in ileal ligated loop, phosphorylable amino compounds such as creatine, L-aspartic and L-glutamic acids also increased calcium absorption; other effective compounds such as D- and L-lysine and D-alanine might be involved in reactions of phorphorylation. L-alanine, L- and D-valine and asparagine were ineffective in enhancing calcium absorption and were not phosphorylable. Injection of creatine into ileal loop induced the formation of its phosphorylated derivative. Absorption of the amino compound was not correlated with the fact that they were effective on calcium absorption.     

Reevaluation of hydropathy profiles of voltage-gated ionic channels

A reevaluation of the secondary structure of Na, Ca and K channel proteins led to the following results. Only three segments (S1, S5 and S6) of each repeat are sufficiently hydrophobic to be predicted as transmembrane helices, if a window of 19 amino acids is used. Some of the S2 and S3 segments show higher hydrophobic values when calculated with the window of 9 amino acids and can be predicted as short helices. S4 segments are strongly hydrophilic and cannot be predicted as transmembrane helices. Some of the S2, S3 and S4 segments have an amphipathic character; however, these helices do not span a membrane. A model is proposed where 12 hydrophobic transmembrane helices surround 12 shorter helices, forming a hydrophilic pore. In addition, a unique pattern for S4 segments of voltage-gated channel proteins is defined.     

Stimulation of sporulation by ppApp in a conditionally asporogenous rifampin-resistant mutant in Bacillus subtilis

Addition of ppApp to Sterlini-Mandelstam medium stimulates sporulation of a conditionally asporogenous rifampin-resistant mutant of Bacillus subtilis to the same extent as the effect of 4 amino acids. Mutant cells sporulating in the presence of amino acids also produce 2 phosphorylated nucleotides one of which comigrated with ppApp on PEI thin layer chromatogram.